Disturbances in ion channel function is a fundamental aspect of the arrhythmias of sudden cardiac death. Recently, there has been is increased appreciation that cardiac ion channels have very short half lives, and much of disturbed ion channel function in disease results from altered trafficking and altered localization to the appropriate subdomain. Regulation of intracellular trafficking remains poorly understood for cardiac ion channels. In previous studies, we have identified how Connexin43 (Cx43) gap junction hemichannels undergo cytoskeleton based Targeted Delivery to cardiac intercalated discs. Recently, we found an endogenous truncated isoform of Cx43, the alternatively translated GJA1-20k, which is essential to full length Cx43 forward trafficking and can rescue ischemia induced defects in Cx43 gap junction localization. Our studies reveal that GJA1-20k is a critical molecule for channel and organelle movement. Understanding the molecular details of its role in cardiac muscle cells will reveal fundamental mechanisms of channel localization and also facilitate translational use of this molecule into novel and clinically important therapeutic strategies. The objective of this application is to explore the molecular mechanisms by which GJA1-20k organizes trafficking highways. Our central hypothesis is that GJA1-20k, which increases during episodes of myocardial stress, is a powerful cytoskeleton actin nucleator that builds the dynamic trafficking highways essential for Cx43 gap junction delivery. Our expertise in ion channel trafficking and identification of GJA1-20k, as well as our cell biological tools and the new mouse model we generated for this proposal, allow us to successfully carry out the planned experiments by pursuing two specific aims: 1) Is GJA1-20k essential to the formation of Cx43 gap junctions in vivo? 2) Is GJA1-20k an actin nucleator, capable of patterning cytoskeleton pathways for channel delivery? Once successfully completed, the expected outcomes are to identify that GJA1-20k is a vital cardiomyocyte protein whose mechanism of action is inherently linked to the actin and microtubule cytoskeleton, is important in baseline healthy heart, and essential during times of stress. Such results are expected to have a positive impact because we will have introduced a new therapeutic approach (exogenous GJA1-20k) to limit pathologic trafficking in arrhythmogenic disease. At the same time, and perhaps of even more long-term consequence, the studies will help resolve a fundamental unsolved mystery in the biology of protein trafficking which is how membrane proteins are delivered directly to their respective subdomain.